The focus of this supplement is to determine if cathepsin K is essential for proper dental enamel formation. The original application (R01 DE016276) focused on defining how enamelysin (matrix metalloproteinase-20;MMP20) processes enamel proteins and focused on determining how such processing allows for proper enamel development. This supplemental application extends and enhances the original studies because it is focused on identifying the function of a protease (cathepsin K) that is well known for its role in bone remodeling, but was heretofore not known as expressed by enamel producing cells (ameloblasts). During amelogenesis, enamel matrix proteins are degraded and removed to generate the virtually protein-free mineral of mature enamel. Proteolytic processing is critical for enamel formation as homozygous mutation of either of the resident enamel proteases, MMP20 or kallikrein-4 (KLK4), results in defective hypomineralized enamel. The long-term goal of our research is to achieve a better understanding of protein degradation and removal as dental enamel attains its final hardened form. In this supplemental application we provide preliminary data demonstrating that cathepsin K is expressed by ameloblasts and that its expression increases as enamel development progresses. We also show that cathepsin K rapidly degrades recombinant amelogenin in vitro. Notably, we discovered that cathepsin K null mice have significantly reduced enamel hardness when compared to controls. Thus, we hypothesize that cathepsin K actively participates in the degradation of enamel matrix proteins that are resorbed by maturation stage ameloblasts. The overall objective of this proposal is to determine if cathepsin K is an essential protease for proper dental enamel development. To this end, we will identify the location of total and active cathepsin K within the developing tooth (Aim 1). We will localize cathepsin K in mouse incisors by use of immunogold labeling and will identify the location of active cathepsin K by use of a catalytic histochemical assay. In Aim 2 we will determine if enamel development is altered in the cathepsin K null mouse. Tooth morphology, volume, and density will be examined by radiography and microCT analysis. Enamel crystal structure and mineral surface characteristics will be characterized by nanoindentation and SEM. Results from these studies will accomplish our overall objective of understanding if, and/or to what extent, cathepsin K plays a role in enamel development. We will also further our knowledge of how enamel starts as a protein rich tissue, but ends as a virtually protein-free mineralized hard tissue. Relevance to public health: These studies will lead to a better understanding of how dental enamel forms on teeth. This knowledge is a necessary first step to eventually understand how to treat individuals with genetically malformed dental enamel. At the outset, the genes required for enamel formation should be known and well characterized. These studies are also necessary to enhance the possibility that tissue engineering techniques will eventually provide therapeutic interventions. PUBLIC HEALTH RELEVANCE: Little is known about how enamel matrix proteins are degraded and removed from maturing dental enamel. We show that the cells (ameloblasts) responsible for enamel formation, express an enzyme (cathepsin K) that degrades proteins. This proposal seeks to demonstrate that cathepsin K plays an essential role in enamel formation by degrading enamel matrix proteins as the proteins are removed from the maturing enamel.